EP1142994B1

EP1142994B1 - Thermostable creatine amidinohydrolase and process for producing the same

Info

Publication number: EP1142994B1
Application number: EP99961487A
Authority: EP
Inventors: Keisuke Furukawa; Yasuji Koyama; Masaru Suzuki
Original assignee: Kikkoman Corp
Current assignee: Kikkoman Corp
Priority date: 1999-01-01
Filing date: 1999-12-28
Publication date: 2007-08-15
Anticipated expiration: 2019-12-28
Also published as: JP3773160B2; EP1142994A1; WO2000040708A1; JP2000201675A; DE69936880T2; US6821766B1; DE69936880D1; EP1142994A4

Abstract

A thermostable creatine amidinohydrolase having the following physicochemical properties: (a) hydrolyzing 1 mol of creatine to give 1 mol of sarcosine and 1 mol of urea; (b) having a substrate specificity to creatine; (c) having an optimum pH range of 7.0 to 8.0; (d) having a stable pH range of 4.0 to 11.0; (e) having the optimum function temperature of around 45 °C; (f) being stable at 53 °C; and (g) having a molecular weight of 92,000 Da (determined by gel filtration method).

Description

The present invention relates to a thermostable creatine amidinohydrolase and a process for producing the same.
Creatine amidinohydrolase is an enzyme catalyzing hydrolysis of creatine to give sarcosine and urea, which can be used to determine an amount of creatine in human serum or urine, being usable as a diagnostic agent for various diseases such as kidney diseases and the like.
For the reason that a creatine amidinohydrolase from Alcaligenes having Km value of approximately 13 mM and thermostability of around 45°C shows a lower Km value, and moreover, taking into consideration the distribution and the sales of the agent in liquid form, a creatine amidinohydrolase having stability at an increased temperature has been sought. Furthermore, conventionally, attempts to improve the thermostability of creatine amidinohydrolase by genetic modification frequently ended with the results showing the same Km value as an original strain or even a decreased value, and there have been no cases where the thermostability and the Km value thereof were improved.
The object of the present invention is to provide a thermostable creatine amidinohydrolase and a process for producing the same.
The present inventors have further studied to solve the above problem, and found that a thermostable creatine amidinohydrolase was obtainable by modifying a gene of an Alcaligenes creatine amidinohydrolase ( JP-A-8-89255 (1996 )), thus completing the present invention.
Specifically, the present invention provides an isolated thermostable creatine amidinohydrolase obtainable from E. coli JM109 (pUCE100 B40) (Accession Number FERM BP-6867) having the following physicochemical properties:

(a) hydrolyzing 1 mol of creatine to give 1 mol of sarcosine and 1 mol of urea;
(b) having a substrate specificity to creatine;
(c) having an optimum pH range of 7.0 to 8.0;
(d) having a stable pH range of 4.0 to 11.0;
(e) having the optimum temperature of around 45°C;
(f) being thermostable at 53°C; and
(g) having a molecular weight of 92,000 Da (as determined by gel filtration).

In another aspect, the invention provides a process for producing the thermostable creatine amidinohydrolase which comprises incubating a microorganism capable of producing the above amidinohydrolase, and recovering said amidinohydrolase from the culture,
wherein said microorganism is an E.coli JM109 strain (pUCE100 B-40) (Accession Number: FERM BP-6867)

Fig.1 shows the optimum pH of the enzyme of the invention.
Fig.2 shows the optimum temperature range of the enzyme of the invention.
Fig.3 shows the stable pH range of the enzyme of the invention.
Fig.4 shows the thermostability of the enzyme of the invention.

The present invention will be described in detail below.
The creatine amidinohydrolase can be obtained, for example, as follows.
In one method, a mutant can be produced artificially by exposing an Alcaligenes bacterium to radiation such as a UV light or by treating said bacterium with chemical mutagens (such as nitrous acid, hydroxylamine, etc.) to obtain mutants and selecting a mutant having the thermostable creatine amidinohydrolase activity of the present invention from the obtained mutants according to a method for determining enzyme properties such as the method mentioned below. The mutant with the activity of interest is incubated in a suitable medium to collect the thermostable creatine amidinohydrolase produced thereby
In an alternative method, DNA encoding the thermostable creatine amidinohydrolase can be obtained from an Alcaligenes bacterium or a mutant strain thereof according to usual methods, or DNA encoding the creatine amidinohydrolase can be mutated into DNA encoding the thermostable creatine amidinohydrolase with mutagenesis treatment.
For example, DNA of interest can be obtained as follows.
Genomic DNA or mRNA is taken from the above bacterium, and in the case of genomic DNA, said DNA is cut into an appropriate length to incorporate it into a suitable vector to create a genomic DNA library, while in the case of mRNA, cDNA library is created with reverse transcriptase, followed by performance of an immunological reaction with an anti-creatine amidinohydrolase antibody, hybridization with a labeled probe with specific sequence from a known gene sequence for creatine amidinohydrolase, or polymerase chain reaction (PCR) using primers with said specific sequence, whereby DNA or cDNA containing the creatine amidinohydrolase gene can be selected or selectively amplified. If necessary, said gene can be incorporated into a vector to be transformed into a bacterial host, which is then incubated thereby propagating the gene of interest. Furthermore, if required, a restriction map of the obtained gene is made, and this gene is cleaved to give a fragment containing the creatine amidinohydrolase gene utilizing the restriction map. Thus, incorporating the obtained gene into a suitable vector (preferably plasmid), and applying mutagenesis thereto as mentioned below enables the DNA encoding the thermostable creatine amidinohydrolase of interest to be obtained.
Alternatively, if a known transformant containing a creatine amidinohydrolase gene is available, a vector (particularly a plasmid) containing the gene of interest can be collected therefrom according to usual methods. For example, the plasmid containing the gene of interest can be extracted and purified from E.coli JM109 (pUCE 100) (which has been deposited under the Budapest Treaty with National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (1-1-3, Higashi, Tsukuba, Ibaragi, Japan) under Accession Number: FERM BP-4803) which retains a recombinant plasmid pUCE 100 DNA ( JP-A-8-89255 (1996 )) containing a creatine amidinohydrolase gene from Alkaligenes sp. KS-85 strain which encodes an amino acid sequence of SEQ ID NO:1 by using QIAGEN (Funakoshi, Japan) or the like. DNA encoding the thermostable creatine amidinohydrolase of interest can be obtained by mutagenesis as mentioned below of the obtained plasmid.
Vector DNA usable in the invention includes a bacteriophage vector DNA, a plasmid vector DNA, etc. To be specific, vectors such as pUC118 (Takara Shuzo, Japan) and pBluescript (Stratagene, U.S.A.) are preferred.
A method for obtaining a thermostable creatine amidinohydrolase gene by a mutagenesis treatment of the recombinant plasmid containing DNA which encodes the creatine amidinohydrolase as obtained in the above procedures includes, but is not limited to, point mutation where the recombinant plasmid DNA is mutated at random with a chemical mutagen such as hydroxylamine or nitrous acid or by PCR, and site-specific mutagenesis, the technique being known to cause site-specific mutations comprising substitutions or deletions using a commercially available kit; a method where the above-mentioned recombinant plasmid DNA is selectively cleaved and the cleaved DNA is connected after deletion or addition of a selected oligonucleotide; and an oligonucleotide mutagenesis method. As for a mutagenesis method by genetic engineering, reference can be made to J. Sambrook et al., Molecular Cloning A Laboratory Manual (Second ed.), Cold Spring Harbor Laboratory Press, 1989, for example. Thus, the plasmid which contains DNA encoding a protein consisting of an amino acid sequence comprising mutations (i.e. deletions, substitutions, insertions, or additions of one or more amino acid residues) relative to an amino acid sequence of e.g. SEQ ID NO:1, and having thermostable creatine amidinohydrolase activity, can be obtained.
The recombinant DNA treated as above can be purified using a desalting column, QIAGEN (Funakoshi, Japan) or the like to obtain various recombinant DNAs.
Using thus obtained DNAs, E.coli K12, preferably E.coli JM 109 (TOYOBO, Japan), or XL1-Blue (Funakoshi, Japan) or the like can be transformed or transduced to obtain transformed or transduced microorganisms which contain recombinant DNAs carrying different creatine amidinohydrolase gene fragments.
In the case of the transformed microorganisms, for example, the following method can be adopted to obtain a strain which produces the thermostable creatine amidinohydrolase having desired properties (being thermostable and showing a decreased Km value) from the obtained transformants (in which the recombinant plasmid DNAs comprising different thermostable creatine amidinohydrolase genes are contained).
At first, each colony of the above obtained transformants is incubated in a liquid TY mecium (with addition of 50 µg Ampicilin and 1mM IPTG) to inductively produce various kinds of thermostable creatine amidinohydrolase encoded in the recombinant plasmid DNAs. After incubation, the obtained culture is subjected to ultrasonic disintegration and extraction to heat-treat the crude enzyme extract for 30 min at 50°C, followed by measurement of the remaining activity and the Km value by Lineweaver-Burk plot. Furthermore, the measured results of each mutant are compared to those of a wild type protein which has been extracted, treated, and measured in the same manner, thereby selecting the transformant of interest.
To produce a thermostable creatine amidinohydrolase using the above obtained transformed or transduced microorganisms capable of producing the thermostable creatine amidinohydrolase, preferably using a strain belonging to the genus Escherichia, the following method can be employed.
The above microorganism may be incubated by a common solid culture method, but the application of a liquid culture method is preferred.
A medium usable for incubating the above microorganism comprises at least one nitrogen source such as yeast extract, peptone, meat extract, corn steep liquor, the effusion of soy bean or wheat koji, etc., with addition of at least one inorganic salt such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, ferric chloride, ferric sulfate or manganese sulfate, and optionally, saccharide materials, vitamins, etc.
Adjustment of the initial pH of the medium to 7-9 is appropriate. Incubation is preferably performed at 30°C-42°C, preferably around 37°C for 6-24 hours by submerged aeration-agitation culture, shake culture, static culture, etc. After incubation, usual methods for collecting enzymes can be used to recover the thermostable creatine amidinohydrolase from the culture.
Cells are separated from the culture by subjecting it to such as filtration, centrifugation, etc., and then washed. The thermostable creatine amidinohydrolase is preferably collected from the cells. In such a case, the cells may be used as they are, however, it is preferred to collect the creatine amidinohydrolase therefrom by the following method; a method wherein cells are disintegrated with various disintegrators such as ultrasonic disintegrator, French press, dynamill, etc.; a method wherein the cell wall of the cells is lysed with such an enzyme as lysozyme and; or a method wherein an enzyme is extracted from the cells with a surfactant such as TritonX-100.
To isolate the thermostable creatine amidinohydrolase from the thus-obtained crude enzyme solution, usual methods for purifying an enzyme can be used. For example, a suitable combination of ammonium sulfate salting-out, organic solvent precipitation, ion-change chromatography, gel filtration chromatography, adsorption chromatography, electrophoresis, etc. is preferred.
The physicochemical properties of the obtained thermostable creatine amidinohydrolase are as follows.

(1) Action: The enzyme hydrolyzes 1 mol of creatine to give 1 mol of sarcosine and 1 mol of urea;
(2) Substrate specificity: The enzyme has a substrate specificity to creatine;
(3) Optimum pH: The enzyme reaction at 37°C for 10 min at each pH using the following buffers: 50mM sodium acetate-acetic acid buffer (pH 4.0-5.5), 50mM MES buffer (pH5.5-6.5), 50mM phosphate buffer (pH 6.5-8.0), 50mM Tris-HCl buffer (pH 8.0-9.0), 50mM CHES buffer (pH 9.0-10.0), and 50mM CAPS buffer (pH 10.0-11.0), indicates the relative activity shown in Fig.1. As seen therefrom, the optimum pH of the enzyme is 7.0-8.0.
(4) Optimum temperature: Enzyme activity measured at different temperatures using a reaction solution with the same composition as the one used in the below-mentioned assay is shown in Fig.2. As seen therefrom, the optimum temperature is around 45°C.
(5) Stable pH range: Remaining activity of the enzyme determined after treatment at 25°C for 17 hours at pH 4.0-11.0 using the following buffer solutions: 50mM sodium acetate-acetic acid buffer (pH 4.0-5.5), 50mM MES buffer (pH5.5-6.5), 50mM phosphate buffer (pH 6.5-8.0), 50mM Tris-HCl buffer (pH 8.0-9.0), 50mM CHES buffer (pH 9.0-10.0), and 50mM CAPS buffer (pH 10.0-11.0), is shown in Fig.3. As seen therefrom, the stable pH range is pH4.0-11.0.
(6) Thermostability: Thermostability measured after treatment at each temperature for 30 min using 50mM Tris-HCl buffer (pH 7.5) is shown in Fig.4. The enzyme of the invention is stable up to approximately 53°C.
(7) Assay for measuring enzyme activity: Enzyme activity is measured under the following conditions. One unit used herein indicates the enzyme activity which generates 1 µ mol of urine per minute.

(Preparation of reagents)

The first solution: substrate solution

6.63g of creatine is dissolved in 500ml of 50mM phosphate buffer (pH7.7).

The second solution: color-development solution

10g of p-dimethylaminobenzaldehyde is dissolved in 500ml of special grade ethanol,

(Measuring procedure)

1) Preincubation of 0.9ml of the first solution at 37°C for 5 min.
2) Mixing 0.1ml of an enzyme solution (adjusted to approximately 1-2U/ml) thereto to allow reaction at 37°C for 10 min
3) Mixing 2ml of the above second solution
4) Allowing the mixture to stand at 25°C for 20 min, followed by measurement of the absorbance at 435nm (OD sample)
5) Blank value was measured by: incubating 0.9ml of the first solution at 37°C for 10min; mixing 2ml of the above second solution thereto; further mixing 0.1ml of the enzyme solution thereto; allowing to stand the mixture at 25°C for 20 min; and measuring the absorbance at 435nm (ODblank)

(Calculation of activity)

$U / ml = ΔOD \times 18.06 \times dilution rate$
Herein, ΔOD = ODsample-ODblank, and 18.06 is a coefficient calculated from a calibration curve of urine.

(8) Km value: The Km value of the enzyme measured with the above-mentioned assay method was 8.6mM (for creatine) as determined from Lineweaver-Burk plot.
(9) Molecular weight: 92,000 Da (determined by gel filtration method).

Embodiments of the invention will be described below by means of illustration, but it is not intended that the scope of the invention is limited to these.

Example 1

(1) Preparation of a recombinant plasmid pUCE100DNA

E.coli JM109(pUCE100)(FERM BP-4803) was inoculated in 20ml of TY medium (1% bacto-tryptone, 0.5% peptone, 0.25% NaCl), which was incubated at 37°C for 18 hours while shaking, thereby obtaining the culture. The culture was then subjected to centrifugation at 6000rpm for 10 min to harvest cells. A recombinant plasmid pUCE100 DNA was extracted therefrom and purified with QIAGEN tip-100 to obtain 70 µ g of the DNA.

(2) Mutagenesis

XL1-RED (STRATAGENE) (upon proliferation, errors in plasmid replication occur readily, resulting in mutagenesis) is transformed with 2 µg out of 100 µg of the above recombinant plasmid DNA according to the method of D.M.Morrison (Method in Enzymology, 68, 326-331, 1979) to obtain approximately 1500 transformants. Among them, 500 colonies were all inoculated in 20ml of the TY medium and incubated at 37°C for 18 hours while shaking. After incubation, the culture was subjected to centrifugation at 6000rpm for 10 min, to harvest cells. The plasmid pUCE100 was extracted therefrom and purified with QIAGEN tip-100 (Funakoshi, Japan) to obtain 70 µ g of a mutated recombinant plasmid pUCE100DNA. E.coli JM109 strain (TOYOBO, Japan) was transformed using 5 µ g of this plasmid to obtain approximately 2000 transformants retaining the mutated plasmid.

(3) Screening of a mutant having the improved thermostability and reactivity to a substrate

At first, each colony of the transformants obtained in the above is incubated in 2ml TY medium (with addition of 50 µg Ampicilin and 1mM IPTG), thereby allowing the induction of the various thermostable creatine amidinohydrolase genes contained in the plasmid. After incubation, the obtained culture was subjected to ultrasonic disintegration and extraction, and the crude enzyme extract was heat-treated at 50°C for 30 min to determine the remaining activity; and the Km value was measured by Lineweaver-Burk plot with regard to the enzymes with good thermostability. Further, the measured result of each mutant was compared with that of a wild type creatine amidinohydrolase which was extracted and measured in the same manner to select a mutant being adequate for the purpose, thus the thermostable creatine amidinohydrolase was obtained from a mutant, E.coli JM109 (pUCE100 B-40).
E.coli JM109(pUCE100 B-40) has been deposited under the Budapest Treaty with National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (1-1-3, Higashi, Tsukuba, Ibaragi, Japan) under the accession number of FERM BP-6867. (This microorganism deposit was transferred as an international deposit on September 2, 1999, from the national deposit FERM P-15971 deposited on November 28, 1996.)

Example 2

Collection of the thermostable creatine amidinohydrolase enzyme

The mutant, E.coli JM109 (pUCE100 B-40) obtained in Example 1 was incubated for 16 hours while shaking in a Sakaguchi flask containing 100ml of TY medium (1% bacto-tryptone, 0.5% bacto-yeast extract, 0.5% NaCl, pH7.5) supplemented with 1mM of isopropyl- β -D-galactoside, which was then flame-seeded in a 30L fermenter containing 20L of TY medium being prepared in the same manner. After seeding, it was incubated at 37°C at 450 rpm for 20 hours under aeration of 20L/min.
After incubation, the cells were harvested from 20L of the culture mixture using Microza (Asahi Chemical Industry, Japan, PW-303), and washed with 20mM phosphate buffer (pH7.5), then suspended in 10L of the same buffer.

Step 1 (Preparation of the crude enzyme solution):

20g of lysozyme (in 50mM phosphate buffer, pH7.5, 100ml) and 1L of 0.55M EDTA, pH8.0 were added to the above cell suspension (10L) and mixed, which was allowed to stand at 30°C overnight, then treated to remove nucleic acids by adding dropwise 500ml of 5% protamine solution in water (pH8.0) with stirring. This solution was dialyzed against 10mM CAPS-NaOH buffer (pH10.0) (referred to as buffer A hereinafter).

Step 2 (DEAE-cellulose treatment):

Approximately 3kg (by wet weight) of cellulose was added to the dialysate solution (approximately 28L) and mixed, thereby adsorbing the thermostable creatine amidinohydrollase thereto, then the DEAE-cellulose was washed in buffer A containing 5% glycerine and 0.05%2-mercaptoethanol; and the thermostable creatine amidinohydrolase was eluted with buffer A containing 0.5M KCl, followed by ultrafiltration.

Step 3 (DEAE-Sepharose CL-4B (Pharmacia) treatment):

Approximately 1.0kg (by wet weight) of DEAE-Sepharose CL-4B which was buffered in buffer A was added to the condensate (approximately 1.0L) from step 2 and mixed, thereby adsorbing the thermostable creatine amidinohydrolase thereto; then the DEAE-Sepharose CL-4B was washed in buffer A containing 0.05M KCl; and the thermostable creatine amidinohydrolase was eluted with buffer A, followed by ultrafiltration.

Step 4 (Sephacryl S-200 (Pharmacia) treatment:

The condensate (approximately 1.0L) was molecular-sieved on Sephacryl S-200 filled in a column to collect 2.2g of the active fraction. The specific activity of the fraction was 9U/OD280nm. The properties of the obtained enzyme were the same as those mentioned in the above.
The present invention elucidated that the thermostable creatine amidinohydrolase can be produced efficiently.
While the invention can be carried out by those skilled in the art with various modifications or variations within the scope of the invention described in the attached claims or equivalents thereof, such modifications or variations are also intended to be included in the present invention.
All publications and patent applications cited herein are incorporated herein by reference in their entirety.

SEQUENCE LISTING

<110> KIKKOMAN CORPORATION
<120> THERMOSTABLE CREATINE AMIDINOHYDROLASE AND PROCESS FOR PRODUCING THE SAME
<130> PH-698-PCT
<140>
<141>
<150> JP 11-033359
<151> 1999-01-01
<160> 1
<210> 1
<211> 404
<212> PRT
<213> Alcaligenes sp.
<400> 1

Claims

An isolated thermostable creatine amidinohydrolase obtainable from E. coli JM109 (pUCE100 B-40) (Accession Number FERM BP-6867) which has the following physicochemical properties:
(a) hydrolyzing 1 mol of creatine to give 1 mol of sarcosine and I mol of urea;

(b) having a substrate specificity to creatine;

(c) having an optimum pH of 7.0 to 8.0;

(d) having a stable pH range of 4.0 to 11.0;

(e) having the optimum temperature of around 45°C;

(f) being thermostable at 53°C; and

(g) having a molecular weight of 92,000 Da (as determined by gel filtration).
A process for producing a thermostable creatine amidinohydrolase comprising: incubating a microorganism capable of producing the thermostable creatine amidinohydrolase of claim 1 and recovering said thermostable creatine amidinohydrolase from the culture wherein said microorganism is E. coli JM109 (pUCE100 B-40) (Accession Number FERM BP-6867).